Solubility optimization of immunobinders

ABSTRACT

The invention provides methods of using sequence based analysis and rational strategies to improve the solubility of immunobinders, and in particular of single chain antibodies (scFvs). The invention provides methods of engineering immunobinders, and in particular scFvs, by performing one or more substitutions with hydrophilic residues identified by analysis of a database of selected, stable scFv sequences. The invention also provides immunobinders with optimized solubility prepared according to the engineering methods of the invention.

RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.15/378,649 filed Dec. 14, 2016 (now Allowed), which is a divisional ofU.S. application Ser. No. 13/000,460, a 371 application filed Dec. 21,2010 (now U.S. Pat. No. 9,556,265), which claims priority fromPCT/CH2009/000221, filed Jun. 25, 2009, which claims priority of U.S.61/075,692, filed Jun. 25, 2008.

BACKGROUND OF THE INVENTION

Antibodies have proven to be very effective and successful therapeuticagents in the treatment of cancer, autoimmune diseases and otherdisorders. While full-length antibodies typically have been usedclinically, there are a number of advantages that use of an antibodyfragment can provide, such as increased tissue penetration, absence ofFc-effector function combined with the ability to add other effectorfunctions and the likelihood of less systemic side effects resultingfrom a shorter in vivo half life systemically. The pharmacokineticproperties of antibody fragments indicate that they may be particularlywell suited for local therapeutic approaches. Furthermore, antibodyfragments can be easier to produce than full-length antibodies incertain expression systems.

One type of antibody fragment is a single chain antibody (scFv), whichis composed of a heavy chain variable domain (V_(H)) conjugated to alight chain variable domain (V_(L)) via a linker sequence. Thus, scFvslack all antibody constant region domains and the amino acid residues ofthe former variable/constant domain interface (interfacial residues)become solvent exposed. An scFv can be prepared from a full-lengthantibody (e.g., IgG molecule) through established recombinantengineering techniques. The transformation of a full length antibodyinto an scFv, however, often results in poor stability and solubility ofthe protein, low production yields and a high tendency to aggregate,which raises the risk of immunogenicity.

Accordingly, attempts have been made to improve properties such assolubility of scFvs. For example, Nieba, L. et al. (Prot. Eng. (1997)10:435-444) selected three amino acid residues known to be interfacialresidues and mutated them. They observed increased periplasmicexpression of the mutated scFv in bacteria, as well as a decreased rateof thermally induced aggregation, although thermodynamic stability andsolubility were not significantly altered. Moreover, in theirpublication, they expressively state they did not observe any solubilityimprovement of the native protein state of the engineered scFvs asdetermined by the PEG precipitation method. Other studies in which sitedirected mutagenesis was carried out on particular amino acid residueswithin the scFv also have been reported (see e.g., Tan, P. H. et al.(1988) Biophys. J. 75:1473-1482; Worn, A. and Pluckthun, A. (1998)Biochem. 37:13120-13127; Worn, A. and Pluckthun, A. (1999) Biochem.38:8739-8750). In these various studies, the amino acid residuesselected for mutagenesis were chosen based on their known positionswithin the scFv structure (e.g., from molecular modeling studies).

In another approach, the complementarity determining regions (CDRs) froma very poorly expressed scFv were grafted into the framework regions ofan scFv that had been demonstrated to have favorable properties (Jung,S. and Pluckthun, A. (1997) Prot. Eng. 10:959-966). The resultant scFvshowed improved soluble expression and thermodynamic stability.

Progress in the engineering of scFvs to improve solubility and otherfunctional properties is reviewed in, for example, Worn, A. andPluckthun, A. (2001) J. Mol. Biol. 305:989-1010. New approaches,however, are still needed that allow for rational design ofimmunobinders, in particular of scFvs with superior solubility.Moreover, methods of engineering scFvs, and other types of antibodies,to thereby impart improved solubility—especially solubility of thenative protein—, are still needed.

SUMMARY OF THE INVENTION

This invention provides an immunobinder comprising a solubilityenhancing motif in the variable heavy chain region V_(H) as well asmethods of engineering immunobinders, such as scFv antibodies, to conferimproved solubility. In particular embodiments, the methods of theinvention comprise the substitution of amino acids within a sequence ofthe variable heavy chain region and/or the variable light chain regionof an immunobinder that are potentially problematic for solubility withpreferred amino acid residues that confer improved solubility. Forexample, in certain preferred embodiments, a hydrophobic residue issubstituted with a hydrophilic residue.

Preferably, the provided immunobinder, the immunobinder used in, orproduced by, the engineering methods of the invention is an scFv, butother immunobinders, such as full-length immunoglobulins, Fab fragments,single domain antibodies (e.g., Dabs) and Nanobodies also can beengineered according to the method. The invention also encompassesimmunobinders prepared according to the engineering method, as well ascompositions comprising the immunobinders and a pharmaceuticallyacceptable carrier.

In one aspect, the invention provides an immunobinder comprising one ofthe following solubility enhancing motifs in the heavy chain amino acidpositions 12, 103 and 144 (AHo numbering):

(a) Serine (S) at heavy chain amino acid position 12;

(b) Serine (S) at heavy chain amino acid position 103; and

(c) Threonine (T) at heavy chain amino acid position 144; or

(a1) Serine (S) at heavy chain amino acid position 12;

(b1) Threonine (T) at heavy chain amino acid position 103; and

(c1) Serine (S) at heavy chain amino acid position 144; or

(a2) Serine (S) at heavy chain amino acid position 12;

(b2) Threonine (T) at heavy chain amino acid position 103; and

(c2) Threonine (T) at heavy chain amino acid position 144; or

(a3) Serine (S) at heavy chain amino acid position 12;

(b3) Serine (S) at heavy chain amino acid position 103; and

(c3) Serine (S) at heavy chain amino acid position 144.

In another aspect, the invention provides a method of engineering animmunobinder, the immunobinder comprising (i) a heavy chain variableregion, or fragment thereof, the heavy chain variable region comprisingV_(H) framework residues and/or (ii) a light chain variable region, orfragment thereof, the light chain variable region comprising V_(L)framework residues, the method comprising:

A) selecting at least two amino acid positions within the V_(H)framework residues, the V_(L) framework residues or the V_(H) and V_(L)framework residues for mutation; and

B) mutating the at least two amino acid positions selected for mutation,

wherein if the at least two amino acid positions selected for mutationare within the V_(H) framework residues, the substitution is at one ormore heavy chain amino acid positions selected from the group consisting12, 103 and 144 (according to AHo numbering convention) and/or

wherein if the one or more amino acid positions selected for mutationare within the V_(L) framework residues, the substitution is at a lightchain amino acid position selected from the group consisting of 15, 52and 147 (according to AHo numbering convention; amino acid positions 15,44 and 106 using Kabat numbering).

The at least two amino acid positions selected for mutation, and theamino acid residue(s) inserted at the selected position(s) are describedin further detail below. The amino acid position numbering set forthbelow uses the AHo numbering system; the corresponding positions usingthe Kabat numbering system are described further herein and theconversion tables for the AHo and Kabat numbering systems are set forthbelow in the detailed description. The amino acid residues are set forthusing standard one letter abbreviation code.

It has surprisingly been found that the presence of the indicatedmutations at the indicated positions increase the overall solubility ofthe immunobinder without having a negative impact on other functionalproperties of the protein. For instance, in case of the combination ofthe three solubility enhancing mutations V12S, L144S and V103T in theV_(H) of an scFv, it was found that said substitutions account for about60% the entire scFv's solubility. This differs from attempts stated inthe prior art to increase the expression yield of immunobinders. Forexample, U.S. Pat. No. 6,815,540 describes the modification of animmunobinder by decreasing the hydrophobicity in an intra-chaininterdomain interface region. For said purpose, 16 positions in thevariable heavy chain framework were identified which may be individuallysubstituted by one or more amino acids selected from a group of 10 aminoacids. It was found that the expression yield of the generated mutantswas increased. Moreover, the same investigation group published in 1999,i.e. three years after the priority date of the mentioned US patent, apaper (see Jung, S., Honegger, A. and Pluckthun, A. (1997) Prot. Eng.10:959-966) stating that the replacement of hydrophobic surface residuesby more hydrophilic ones has been reported in several studies to improveproduction yield. According to the authors, the increase in productionyield is due to improved kinetic portioning between correct folding andaggregation of misfolded material, while the solubility of the nativeprotein is not even affected, nor its thermodynamic stability in asignificant manner. It is hence clear to the skilled person that thesolubility parameter Plückthun et al refer to concerns only solubleexpression and not the overall solubility of the native protein.

BRIEF DESCRIPTION OF FIGURES

The invention will be better understood and objects other than those setforth above will become apparent when consideration is given to thefollowing detailed description thereof. Such description makes referenceto the annex drawings, wherein:

FIG. 1 depicts the PEG precipitation solubility curves of wild-typeESBA105 (E105) and solubility variants thereof.

FIG. 2 depicts the thermal denaturation profiles for wild-type ESBA105(E105) and solubility variants thereof as measured followingthermochallenge at a broad range of temperatures (25-96° C.).

FIG. 3 depicts an SDS-PAGE gel which shows degradation behavior ofvarious ESBA105 solubility mutants after two weeks of incubation underconditions of thermal stress.

FIG. 4 depicts thermal denaturation curves of EP43max and its optimizedvariants as determined by FTIR analysis.

FIG. 5A and FIG. 5B depicts the thermal stability of 578 min-max and 578min-max_DHP as measured by FT-IR.

FIG. 6A and FIG. 6B illustrate solubility of 578 min-max and 578min-max_DHP as determined by ammonium sulfate precipitation.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains to methods for enhancing the solubility ofimmunobinders. More specifically, the present invention disclosesmethods for optimizing immunobinders by introducing amino acidsubstitutions within the immunobinder that improve the solubility of theimmunobinder. The invention also pertains to engineered immunobinders,e.g., scFvs, produced according to the methods of the invention.

So that the invention may be more readily understood, certain terms arefirst defined. Unless otherwise defined, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, suitablemethods and materials are described below. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

The term “antibody” as used herein is a synonym for “immunoglobulin”.Antibodies according to the present invention may be wholeimmunoglobulins or fragments thereof, comprising at least one variabledomain of an immunoglobulin, such as single variable domains, Fv (SkerraA. and Pluckthun, A. (1988) Science 240:1038-41), scFv (Bird, R. E. etal. (1988) Science 242:423-26; Huston, J. S. et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-83), Fab, (Fab′)2 or other fragments well knownto a person skilled in the art.

The term “antibody framework” or “framework” as used herein refers tothe part of the variable domain, either V_(L) or V_(H), which serves asa scaffold for the antigen binding loops of this variable domain (Kabat,E. A. et al., (1991) Sequences of proteins of immunological interest.NIH Publication 91-3242).

The term “antibody CDR” or “CDR” as used herein refers to thecomplementarity determining regions of the antibody which consist of theantigen binding loops as defined by Kabat E. A. et al., (1991) Sequencesof proteins of immunological interest. NIH Publication 91-3242). Each ofthe two variable domains of an antibody Fv fragment contain, forexample, three CDRs.

The term “single chain antibody” or “scFv” refers to a moleculecomprising an antibody heavy chain variable region (V_(H)) and anantibody light chain variable region (V_(L)) connected by a linker. SuchscFv molecules can have the general structures:NH2-V_(L)-linker-V_(H)-COOH or NH2-V_(H)-linker-V_(L)-COOH.

As used herein, “identity” refers to the sequence matching between twopolypeptides, molecules or between two nucleic acids. When a position inboth of the two compared sequences is occupied by the same base or aminoacid monomer subunit (for instance, if a position in each of the two DNAmolecules is occupied by adenine, or a position in each of twopolypeptides is occupied by a lysine), then the respective molecules areidentical at that position. The “percentage identity” between twosequences is a function of the number of matching positions shared bythe two sequences divided by the number of positions compared×100. Forinstance, if 6 of 10 of the positions in two sequences are matched, thenthe two sequences have 60% identity. By way of example, the DNAsequences CTGACT and CAGGTT share 50% identity (3 of the 6 totalpositions are matched). Generally, a comparison is made when twosequences are aligned to give maximum identity. Such alignment can beprovided using, for instance, the method of Needleman et al. (1970) J.Mol. Biol. 48: 443-453, implemented conveniently by computer programssuch as the Align program (DNAstar, Inc.).

“Similar” sequences are those which, when aligned, share identical andsimilar amino acid residues, where similar residues are conservativesubstitutions for corresponding amino acid residues in an alignedreference sequence. In this regard, a “conservative substitution” of aresidue in a reference sequence is a substitution by a residue that isphysically or functionally similar to the corresponding referenceresidue, e.g., that has a similar size, shape, electric charge, chemicalproperties, including the ability to form covalent or hydrogen bonds, orthe like. Thus, a “conservative substitution modified” sequence is onethat differs from a reference sequence or a wild-type sequence in thatone or more conservative substitutions are present. The “percentagesimilarity” between two sequences is a function of the number ofpositions that contain matching residues or conservative substitutionsshared by the two sequences divided by the number of positions comparedand multiplied by a factor 100. For instance, if 6 of 10 of thepositions in two sequences are matched and 2 of 10 positions containconservative substitutions, then the two sequences have 80% positivesimilarity.

“Amino acid consensus sequence” as used herein refers to an amino acidsequence that can be generated using a matrix of at least two, andpreferably more, aligned amino acid sequences, and allowing for gaps inthe alignment, such that it is possible to determine the most frequentamino acid residue at each position. The consensus sequence is thatsequence which comprises the amino acids which are most frequentlyrepresented at each position. In the event that two or more amino acidsare equally represented at a single position, the consensus sequenceincludes both or all of those amino acids.

The amino acid sequence of a protein can be analyzed at various levels.For example, conservation or variability can be exhibited at the singleresidue level, multiple residue level, multiple residue level with gapsetc. Residues can exhibit conservation of the identical residue or canbe conserved at the class level. Examples of amino acid classes includethe class of amino acids with polar but uncharged side chains or Rgroups (Serine, Threonine, Asparagine and Glutamine); with positivelycharged R groups (Lysine, Arginine, and Histidine); with negativelycharged R groups (Glutamic acid and Aspartic acid); with hydrophobic Rgroups (Alanine, Isoleucine, Leucine, Methionine, Phenylalanine,Tryptophan, Valine and Tyrosine); and the class of special amino acids(Cysteine, Glycine and Proline). Other classes are known to one of skillin the art and may be defined using structural determinations or otherdata to assess substitutability. In that sense, a substitutable aminoacid can refer to any amino acid which can be substituted and maintainfunctional conservation at that position.

It will be recognized, however, that amino acids of the same class mayvary in degree by their biophysical properties. For example, it will berecognized that certain hydrophobic R groups (e.g., Alanine, Serine, orThreonine) are more hydrophilic (i.e., of higher hydrophilicity or lowerhydrophobicity) than other hydrophobic R groups (e.g., Valine orLeucine). Relative hydrophilicity or hydrophobicity can be determinedusing art-recognized methods (see, e.g., Rose et al., Science, 229:834-838 (1985) and Cornette et al., J. Mol. Biol., 195: 659-685 (1987)).

As used herein, when one amino acid sequence (e.g., a first V_(H) orV_(L) sequence) is aligned with one or more additional amino acidsequences (e.g., one or more V_(H) or V_(L) sequences in a database), anamino acid position in one sequence (e.g., the first V_(H) or V_(L)sequence) can be compared to a “corresponding position” in the one ormore additional amino acid sequences. As used herein, the “correspondingposition” represents the equivalent position in the sequence(s) beingcompared when the sequences are optimally aligned, i.e., when thesequences are aligned to achieve the highest percent identity or percentsimilarity.

As used herein, the term “antibody database” refers to a collection oftwo or more antibody amino acid sequences (a “multiplicity” ofsequences), and typically refers to a collection of tens, hundreds oreven thousands of antibody amino acid sequences. An antibody databasecan store amino acid sequences of, for example, a collection of antibodyV_(H) regions, antibody V_(L) regions or both, or can store a collectionof scFv sequences comprised of V_(H) and V_(L) regions. Preferably, thedatabase is stored in a searchable, fixed medium, such as on a computerwithin a searchable computer program. In one embodiment, the antibodydatabase is a database comprising or consisting of germline antibodysequences. In another embodiment, the antibody database is a databasecomprising or consisting of mature (i.e., expressed) antibody sequences(e.g., a Kabat database of mature antibody sequences, e.g., a KBDdatabase). In yet another embodiment, the antibody database comprises orconsists of functionally selected sequences (e.g., sequences selectedfrom a QC assay).

The term “immunobinder” refers to a molecule that contains all or a partof the antigen binding site of an antibody, e.g., all or part of theheavy and/or light chain variable domain, such that the immunobinderspecifically recognizes a target antigen. Non-limiting examples ofimmunobinders include full-length immunoglobulin molecules and scFvs, aswell as antibody fragments, including but not limited to (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fab′ fragment, which is essentially a Fab with part ofthe hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed.1993); (iv) a Fd fragment consisting of the V_(H) and C_(H)1 domains;(v) a Fv fragment comprising the V_(L) and V_(H) domains of a single armof an antibody, (vi) a single domain antibody such as a Dab fragment(Ward et al., (1989) Nature 341:544-546), which consists of a V_(H) orV_(L) domain, a Camelid (see Hamers-Casterman, et al., Nature363:446-448 (1993), and Dumoulin, et al., Protein Science 11:500-515(2002)) or a Shark antibody (e.g., shark Ig-NARs Nanobodies®; and (vii)a nanobody, a heavy chain variable region containing a single variabledomain and two constant domains.

As used herein, the term “functional property” is a property of apolypeptide (e.g., an immunobinder) for which an improvement (e.g.,relative to a conventional polypeptide) is desirable and/or advantageousto one of skill in the art, e.g., in order to improve the manufacturingproperties or therapeutic efficacy of the polypeptide. In oneembodiment, the functional property is stability (e.g., thermalstability). In another embodiment, the functional property is solubility(e.g., under cellular conditions). In yet another embodiment, thefunctional property is aggregation behaviour. In still anotherembodiment, the functional property is protein expression (e.g., in aprokaryotic cell). In yet another embodiment the functional property isthe refolding efficiency following inclusion body solubilization in acorresponding purification process. In certain embodiments, antigenbinding affinity is not a functional property desired for improvement.In still another embodiment, the improvement of the functional propertydoes not involve a substantial change in antigen binding affinity.

The term “solubility” as used herein refers to the solubility of thenative protein, i.e. of the monomeric, non-aggregated and functionalimmunobinder. “Enhanced solubility” means an improvement of thesolubility of the native protein which is preferably determined by aleast one of the following methods: PEG precipitation, ammonium sulphateprecipitation, refolding yield or any other method to determinesolubility that is known to the one skilled in the art. The PEGprecipitation method is a method along the lines as described by Atha etal. in “Mechanism of Precipitation of Proteins by Polyethylene Glycols”,JBC, 256: 12108-12117 (1981). The Ammonium sulphate precipitation cane.g. be carried out as follows: 10 μl aliquots of 20 mg/ml proteinsolutions are prepared to each of which 10 μl of (NH4)₂SO₄ solution of adifferent saturation grade (e.g. 35%, 33%, 31%, 29%, 25%, 20% and 15%)is added, followed by vortexing for 5 seconds and 30 minutes incubationat room temperature. After centrifugation at 6000 rpm at 4° C. for 30minutes, the protein concentration in the supernatant is determined. Inthis method, the comparator for different proteins is the V50 value,which is the percentage of (NH4)₂SO₄ saturation at which 50% percent ofthe protein is precipitated. The V50 is determined from a plot ofsoluble protein determined in the supernatant against the appliedpercentage of (NH4)₂SO₄ saturation. The refolding yield corresponds tothe percentage of correctly folded protein obtained from solubilizedinclusion bodies in a corresponding manufacturing/purification process.The term solubility as used herein does not refer to soluble expression.

Immunobinders with Improved Solubility

In a first aspect, an immunobinder is provided comprising one of thefollowing solubility enhancing motifs in the heavy chain amino acidpositions 12, 103 and 144 (AHo numbering):

(a) Serine (S) at heavy chain amino acid position 12;

(b) Serine (S) at heavy chain amino acid position 103; and

(c) Threonine (T) at heavy chain amino acid position 144; or

(a1) Serine (S) at heavy chain amino acid position 12;

(b1) Threonine (T) at heavy chain amino acid position 103; and

(c1) Serine (S) at heavy chain amino acid position 144; or

(a2) Serine (S) at heavy chain amino acid position 12;

(b2) Threonine (T) at heavy chain amino acid position 103; and

(c2) Threonine (T) at heavy chain amino acid position 144; or

(a3) Serine (S) at heavy chain amino acid position 12;

(b3) Serine (S) at heavy chain amino acid position 103; and

(c3) Serine (S) at heavy chain amino acid position 144; or

It has surprisingly been found that the presence of the indicated aminoacids at the indicated positions increase the overall solubility of theentire immunobinder. For instance, in case of the combination of thethree solubility enhancing mutations V12S, L144S and V103T in the VH ofan scFv, it was found that said substitutions account for about 60% theentire scFv's solubility. Since hydrophobic patches are conserved in thevariable domains of all immunobinders, one or more substitutions at theindicated positions can be used to improve the solubility of anyimmunobinder.

The immunobinder is preferably an scFv antibody, a full-lengthimmunoglobulin, a Fab fragment, a Dab or a Nanobody.

In a preferred embodiment, the immunobinder further comprises one ormore amino acids of the group consisting of (a) Aspartic acid (D) atlight chain amino acid position 31, (b) Glutamic acid (E) at light chainamino acid position 83, (c) Arginine (R) at heavy chain amino acidposition 43, (d) Leucine (L) at heavy chain amino acid position 67 and(e) Alanine (A) at heavy chain amino acid position 78. The presence ofone or more of the indicated amino acids at the corresponding positionsconfers to the immunobinder enhanced stability.

The amino acids indicated herein may be present in the naturallyoccurring immunobinder or derivative thereof, or the immunobinder may beengineered to incorporate one or more of the above mentioned aminoacids.

In a preferred embodiment, the immunobinder disclosed hereinspecifically binds to human TNFα or to human VEGF.

Engineering of Immunobinders with Improved Solubility

As described in detail in the Examples, a sequence-based approachdescribed herein has been used successfully to identify particular aminoacid residue substitutions that confer improved solubility. The Exampleslist exemplary and preferred amino acid substitutions at defined aminoacid positions within the V_(H) regions and optionally in the V_(L)region of an immunobinder (e.g., an scFv). The exemplary substitutionsinclude substitution of problematic amino acid residues (e.g.,solvent-exposed, hydrophobic residues) at amino acid positions that aremore hydrophilic and that occur with greater frequency in the database(e.g., a mature antibody (KDB) database). A particularly preferredsubstitution is the most frequently occurring residue that is morehydrophilic than the problematic residue. In other embodiments, the morehydrophilic amino acid is selected from the group consisting of alanine(A), serine (S), and threonine (T).

Accordingly, the invention provides engineering methods in which one ormore specified amino acid substitutions are introduced into animmunobinder, such as an scFv antibody. Such substitutions can becarried out using standard molecular biology methods, such assite-directed mutagenesis, PCR-mediated mutagenesis and the like.

As set forth in the Examples, the following amino acid positions havebeen identified as problematic amino acids (i.e., so-called “hydrophobicpatches”) for modification in the indicated V_(H) or V_(L) sequences:

V_(H): amino acid positions 2, 4, 5, 12, 103 and 144; and

V_(L): amino acid positions 15, 52 and 147.

The numbering used is the AHo numbering system; conversion tables toconvert the AHo numbering to the Kabat system numbering are set forth asTables 1 and 2.

It has surprisingly been found that substitutions in two or more ofV_(H) positions 12, 103, 144 (according to AHo numbering system) affectthe overall solubility of the entire immunobinder. Since hydrophobicpatches are conserved in the variable domains of all immunobinders, aleast two substitutions at the indicated positions can be used toimprove the solubility of any immunobinder.

In one embodiment, the invention provides a method of engineering animmunobinder, such as an scFv antibody, in which at least two amino acidsubstitutions are made at one or more amino acid positions identifiedsupra to thereby produce variant (i.e., mutated) forms of theimmunobinders.

Thus, in another aspect, the invention provides a method of engineeringan immunobinder, the method comprising:

A) selecting at least two amino acid positions within the V_(H) region,the V_(L) region or the V_(H) and V_(L) regions for mutation; and

B) mutating at least two amino acid positions selected for mutation,

wherein if the at least two amino acid positions selected for mutationare within the V_(H) region, the substitution is at least two heavychain amino acid positions selected from the group consisting of 12, 103and 144 (according to AHo numbering convention; amino acid positions 11,89 and 108 using Kabat numbering), and/or

wherein if the one or more amino acid positions selected for mutationare within the V_(L) region, the substitution is at a light chain aminoacid position selected from the group consisting of 15, 52 and 147(according to AHo numbering convention; amino acid positions 15, 44 and106 using Kabat numbering).

In certain embodiments, the amino acid position is occupied by ahydrophobic amino acid (e.g., Leucine (L) or Valine (V)). In oneembodiment, the amino acid at heavy chain amino acid position 12 isValine (V). In another embodiment, the amino acid at heavy chain aminoacid position 103 is Valine (V). In another embodiment, the amino acidat heavy chain amino acid position 144 is Leucine (L). In anotherembodiment, the amino acid at light chain amino acid position 15 isValine (V). In another embodiment, the amino acid at light chain aminoacid position 52 is Phenylalanine (F). In another embodiment, the aminoacid at light chain amino acid position 147 is Valine (V).

Preferably, the mutating is a substitution of the amino acid at theselected amino acid position with a more hydrophilic amino acid. Inother embodiments, the more hydrophilic amino acid is selected fromserine (S) or threonine (T).

In certain embodiments, the method comprises: a) selecting at least twoamino acid positions within the immunobinder for mutation; and b)mutating the at least two amino acid positions selected for mutation,wherein the mutating comprises at least two substitutions selected fromthe group consisting of:

(i) Serine (S) at heavy chain amino acid position 12 using AHo numbering(position 11 using Kabat numbering);

(ii) Serine (S) or Threonine (T) at heavy chain amino acid position 103using AHo numbering (position 89 using Kabat numbering); and

(iii) Serine (S) or Threonine (T) at heavy chain amino acid position 144using AHo numbering (position 108 using Kabat numbering).

In a much preferred embodiment, at least one of the heavy chain aminoacid positions 12, 103 and 144 is Threonine (T).

In yet other embodiments, the mutating comprises substitution withThreonine (T) a light chain amino acid position 15 using AHo or Kabatnumbering and/or substitution with Alanine (A) at light chain amino acidposition 147 using AHo numbering (position 106 using Kabat numberingconvention).

In other embodiments, the mutating results in at least a 2-fold increasein solubility (e.g., a 2-fold, a 2.5-fold, a 3-fold, a 3.5-fold, a4-fold or greater increase in solubility).

In another embodiment, thermal stability, refolding, expression yield,aggregation and/or binding activity of the immunobinder is not adverselyaffected by the mutating.

In certain embodiments, the mutating further comprises one or morestabilizing mutations at an amino acid position (AHo numberingconvention) selected from the group consisting of: (a) Aspartic acid (D)at light chain amino acid position 31; (b) Glutamic acid (E) at lightchain amino acid position 83; (c) Arginine (R) at heavy chain amino acidposition 43; (d) Leucine (L) at heavy chain amino acid position 67; and(e) Alanine (A) at heavy chain amino acid position 78. These mutationshave proven to have a effect on the stability of the immunobinder.

In another aspect, the invention provides an immunobinder preparedaccording to the method of the invention.

In certain exemplary embodiments, an immunobinder engineered accordingto the method of the invention is an art-recognized immunobinder whichbinds a target antigen of therapeutic importance or an immunobindercomprising variable regions (VL and/or VH regions) or one or more CDRs(e.g., CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3) derived from theimmunobinder of therapeutic importance. For example, immunobinderscurrently approved by the FDA or other regulatory authorities can beengineered according to the methods of the invention. More specifically,these exemplary immunobinders include, but are not limited to, anti-CD3antibodies such as muromonab (Orthoclone® OKT3; Johnson&Johnson,Brunswick, N.J.; see Arakawa et al. J. Biochem, (1996) 120:657-662; Kungand Goldstein et al., Science (1979), 206: 347-349), anti-CD11antibodies such as efalizumab (Raptiva®, Genentech, South San Francisco,Calif.), anti-CD20 antibodies such as rituximab (Rituxan®/Mabthera®,Genentech, South San Francisco, Calif.), tositumomab (Bexxar®,GlaxoSmithKline, London) or ibritumomab (Zevalin®, Biogen Idec,Cambridge Mass.)(see U.S. Pat. Nos. 5,736,137; 6,455,043; and6,682,734), anti-CD25 (IL2Ra) antibodies such as daclizumab (Zenapax®,Roche, Basel, Switzerland) or basiliximab (Simulect®, Novartis, Basel,Switzerland), anti-CD33 antibodies such as gemtuzumab (Mylotarg®, Wyeth,Madison, N.J.—see U.S. Pat. Nos. 5,714,350 and 6,350,861), anti-CD52antibodies such as alemtuzumab (Campath®, Millennium Pharmacueticals,Cambridge, Mass.), anti-GpIIb/gIIa antibodies such as abciximab(ReoPro®, Centocor, Horsham, Pa.), anti-TNFα antibodies such asinfliximab (Remicade®, Centocor, Horsham, Pa.) or adalimumab (Humira®,Abbott, Abbott Park, Ill.—see U.S. Pat. No. 6,258,562), anti-IgEantibodies such as omalizumab (Xolair®, Genentech, South San Francisco,Calif.), anti-RSV antibodies such as palivizumab (Synagis®, Medimmune,Gaithersburg, Md.—see U.S. Pat. No. 5,824,307), anti-EpCAM antibodiessuch as edrecolomab (Panorex®, Centocor), anti-EGFR antibodies such ascetuximab (Erbitux®, Imclone Systems, New York, N.Y.) or panitumumab(Vectibix®, Amgen, Thousand Oaks, Calif.), anti-HER2/neu antibodies suchas trastuzumab (Herceptin®, Genentech), anti-α4 integrin antibodies suchas natalizumab (Tysabri®, Biogenldec), anti-CS antibodies such aseculizumab (Soliris®, Alexion Pharmaceuticals, Chesire, Conn.) andanti-VEGF antibodies such as bevacizumab (Avastin®, Genentech—see U.S.Pat. No. 6,884,879) or ranibizumab (Lucentis®, Genentech).

In certain exemplary embodiments, an immunobinder engineered accordingto the method of the invention is an art-recognized immunobinderdescribed supra. In a preferred embodiment, the immunobinder is an scFvantibody. In other embodiments, the immunobinder is, for example, afull-length immunoglobulin, Dab, Nanobody or a Fab fragment.

Notwithstanding the foregoing, in various embodiments, certainimmunobinders are excluded from being used in the engineering methods ofthe invention and/or are excluded from being the immunobindercomposition produced by the engineering methods. For example, in variousembodiments, there is a proviso that the immunobinder is not any of thescFv antibodies, or variants thereof, as disclosed in PCT PublicationsWO 2006/131013 and WO 2008/006235, such as ESBA105 or variants thereofthat are disclosed in PCT Publications WO 2006/131013 and WO2008/006235, the contents of each of which is expressly incorporatedherein by reference.

Preferably, the immunobinder disclosed herein, used for the methoddisclosed herein or generated by the method disclosed herein,respectively, is an scFv antibody, but other immunobinders, such asfull-length immunoglobulins, Fab fragments or any other type ofimmunobinder described herein (e.g., Dabs or Nanobodies) are alsoencompassed. In a preferred embodiment, the immunobinder as disclosedherein, used for the method disclosed herein or generated by the methoddisclosed herein, respectively, specifically binds to human TNFα or tohuman VEGF.

The invention further encompasses compositions comprising theimmunobinders disclosed herein and a pharmaceutically acceptablecarrier.

ScFv Compositions and Formulations

Another aspect of the invention pertains to scFv composition preparedaccording to the methods of invention. Thus, the invention providesengineered scFv compositions in which one or more solubility-enhancingmutations have been introduced into the amino acid sequence, as comparedto an original scFv of interest, wherein the mutation(s) has beenintroduced into the position of a hydrophobic amino acid residue. In oneembodiment, the scFv has been engineered to contain one mutated aminoacid position (e.g., one framework position). In other embodiments, thescFv has been engineered to contain two, three, four, five, six, seven,eight, nine, ten or more than ten mutated amino acid positions (e.g.,framework positions).

In certain embodiments, the mutating further comprises one or morestabilizing mutations described in PCT Application No.PCT/CH2008/000285, entitled “Methods of Modifying Antibodies, andModified Antibodies with Improved Functional Properties”, filed on Jun.25, 2008 or US Provisional Application No. Ser. No. 61/069,056, entitled“Methods of Modifying Antibodies, and Modified Antibodies with ImprovedFunctional Properties”, filed on Mar. 12, 2008, both of which areincorporated herein by reference. For example the immunobinder mayfurther comprise a substitution at an amino acid position (AHo numberingconvention) selected from the group consisting of: (a) Aspartic acid (D)at light chain amino acid position 31; (b) Glutamic acid (E) at lightchain amino acid position 83; (c) Arginine (R) at heavy chain amino acidposition 43; (d) Leucine (L) at heavy chain amino acid position 67; and(e) Alanine (A) at heavy chain amino acid position 78. One or moremutations at the indicated positions have an effect on the overallstability of the entire immunobinder. In other embodiments, the mutatingfurther comprises the following stabilizing mutations (AHo numberingconvention): (a) Aspartic acid (D) at light chain amino acid position31; (b) Glutamic acid (E) at light chain amino acid position 83; (c)Arginine (R) at heavy chain amino acid position 43; (d) Leucine (L) atheavy chain amino acid position 67; and (e) Alanine (A) at heavy chainamino acid position 78.

Another aspect of the invention pertains to pharmaceutical formulationsof the scFv compositions of the invention. Such formulations typicallycomprise the scFv composition and a pharmaceutically acceptable carrier.As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable for, forexample, intravenous, intramuscular, subcutaneous, parenteral, spinal,epidermal (e.g., by injection or infusion), or topical (e.g., to the eyeor skin) administration. Depending on the route of administration, thescFv may be coated in a material to protect the compound from the actionof acids and other natural conditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents that delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Antibody Position Numbering Systems

Conversion tables are provided for two different numbering systems usedto identify amino acid residue positions in antibody heavy and lightchain variable regions. The Kabat numbering system is described furtherin Kabat et al. (Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). The AHo numbering systemis described further in Honegger, A. and Pluckthun, A. (2001) J. Mol.Biol. 309:657-670).

Heavy Chain Variable Region Numbering

TABLE 1 Conversion table for the residue positions in the Heavy ChainVariable Domain Kabat AHo Kabat AHo Kabat AHo  1 1 44 51 87 101  2 2 4552 88 102  3 3 46 53 89 103  4 4 47 54 90 104  5 5 48 55 91 105  6 6 4956 92 106  7 7 50 57 93 107 * 8 51 58 94 108  8 9 52 59 95 109  9 10 52a 60 96 110 10 11  52b 61 97 111 11 12  52c 62 98 112 12 13 * 63 99113 13 14 53 64 100  114 14 15 54 65 100a 115 15 16 55 66 100b 116 16 1756 67 100c 117 17 18 57 68 100d 118 18 19 58 69 100e 119 19 20 59 70100f  120 20 21 60 71 100g 121 21 22 61 72 100h 122 22 23 62 73 100i 123 23 24 63 74 * 124 24 25 64 75 * 125 25 26 65 76 * 126 26 27 66 77 *127 * 28 67 78 * 128 27 29 68 79 * 129 28 30 69 80 * 130 29 31 70 81 *131 30 32 71 82 * 132 31 33 72 83 * 133 32 34 73 84 * 134 33 35 74 85 *135 34 36 75 86 * 136 35 37 76 87 101  137  35a 38 77 88 102  138  35b39 78 89 103  139 * 40 79 90 104  140 * 41 80 91 105  141 * 42 81 92106  142 36 43 82 93 107  143 37 44  82a 94 108  144 38 45  82b 95 109 145 39 46  82b 96 110  146 40 47 83 97 111  147 41 48 84 98 112  148 4249 85 99 113  149 43 50 86 100 Column 1, Residue position in Rabat'snumbering system. Column 2, Corresponding number in AHo's numberingsystem for the position indicated in column 1. Column 3, Residueposition in Rabat's numbering system. Column 4, Corresponding number inAHo's numbering system for the position indicated in column 3. Column 5,Residue position in Rabat's numbering system. Column 6, Correspondingnumber in AHo's numbering system for the position indicated in column 5

Light Chain Variable Region Numbering

TABLE 2 Conversion table for the residue positions in the Light ChainVariable Domain Kabat AHo Kabat AHo Kabat AHo  1 1 43 51 83 101  2 2 4452 84 102  3 3 45 53 85 103  4 4 46 54 86 104  5 5 47 55 87 105  6 6 4856 88 106  7 7 49 57 89 107  8 8 50 58 90 108  9 9 * 59 91 109 10 10 *60 92 110 11 11 * 61 93 111 12 12 * 62 94 112 13 13 * 63 95 113 14 14 *64  95a 114 15 15 * 65  95b 115 16 16 * 66  95c 116 17 17 51 67  95d 11718 18 52 68  95e 118 19 19 53 69  95f 119 20 20 54 70 * 120 21 21 5571 * 121 22 22 56 72 * 122 23 23 57 73 * 123 24 24 58 74 * 124 25 25 5975 * 125 26 26 60 76 * 126 27 27 61 77 * 127 * 28 62 78 * 128  27a 29 6379 * 129  27b 30 64 80 * 130  27c 31 65 81 * 131  27d 32 66 82 * 132 27e 33 67 83 * 133  27f 34 68 84 * 134 * 35 * 85 * 135 28 36 * 86 * 13629 37 69 87 96 137 30 38 70 88 97 138 31 39 71 89 98 139 32 40 72 90 99140 33 41 73 91 100  141 34 42 74 92 101  142 35 43 75 93 102  143 36 4476 94 103  144 37 45 77 95 104  145 38 46 78 96 105  146 39 47 79 97106  147 40 48 80 98 107  148 41 49 81 99 108  149 42 50 82 100 Column1, Residue position in Rabat's numbering system. Column 2, Correspondingnumber in AHo's numbering system for the position indicated in column 1.Column 3, Residue position in Rabat's numbering system. Column 4,Corresponding number in AHo's numbering system for the positionindicated in column 3. Column 5, Residue position in Rabat's numberingsystem. Column 6, Corresponding number in AHo's numbering system for theposition indicated in column 5

OTHER EMBODIMENTS

It is understood that the invention also includes any of themethodologies, references, and/or compositions set forth in Appendices(A-C) of U.S. Provisional Patent Application Ser. No. 60/905,365(priority giving application of WO 08/110348) and Appendices (A-I) ofU.S. Provisional Patent Application Ser. No. 60/937,112 (priority givingapplication of WO09/000098), including, but not limited to, identifieddatabases, bioinformatics, in silico data manipulation andinterpretation methods, functional assays, preferred sequences,preferred residue(s) positions/alterations, framework identification andselection, framework alterations, CDR alignment and integration, andpreferred alterations/mutations.

Additional information regarding these methodologies and compositionscan be found in U.S. Ser. Nos. 60/819,378; and 60/899,907, and PCTPublication WO 2008/006235, entitled “scFv Antibodies Which PassEpithelial And/Or Endothelial Layers” filed in July, 2006 and Feb. 6,2007 respectively; WO06131013A2 entitled “Stable And Soluble AntibodiesInhibiting TNFα” filed Jun. 6, 2006; EP1506236A2 entitled“Immunoglobulin Frameworks Which Demonstrate Enhanced Stability In TheIntracellular Environment And Methods Of Identifying Same” filed May 21,2003; EP1479694A2 entitled “Intrabodies ScFv with defined framework thatis stable in a reducing environment” filed Dec. 18, 2000; EP1242457B1entitled “Intrabodies With Defined Framework That Is Stable In AReducing Environment And Applications Thereof” filed Dec. 18, 2000;WO03097697A2 entitled “Immunoglobulin Frameworks Which DemonstrateEnhanced Stability In The Intracellular Environment And Methods OfIdentifying Same” filed May 21, 2003; and WO0148017A1 entitled“Intrabodies With Defined Framework That Is Stable In A ReducingEnvironment And Applications Thereof” filed Dec. 18, 2000; and Honeggeret al., J. Mol. Biol. 309:657-670 (2001).

Further, it is understood that the invention also includes methodologiesand compositions suitable for the discovery and/or improvement of otherantibody formats, e.g., full length antibodies or fragments thereof, forexample Fabs, Dabs, and the like. Accordingly, the principles andresidues identified herein as suitable for selection or alteration toachieve desired biophysical and/or therapeutic proprieties that can beapplied to a wide range of immunobinders. In one embodiment,therapeutically relevant antibodies, for example, FDA-approvedantibodies, are improved by modifying one or more residue positions asdisclosed herein.

The invention is not limited to the engineering of immunobinders,however. For example, one skilled in the art will recognize that themethods of the invention can be applied to the engineering of other,non-immunoglobulin, binding molecules, including, but not limited to,fibronectin binding molecules such as Adnectins (see WO 01/64942 andU.S. Pat. Nos. 6,673,901, 6,703,199, 7,078,490, and 7,119,171),Affibodies (see e.g., U.S. Pat. Nos. 6,740,734 and 6,602,977 and in WO00/63243), Anticalins (also known as lipocalins) (see WO99/16873 and WO05/019254), A domain proteins (see WO 02/088171 and WO 04/044011) andankyrin repeat proteins such as Darpins or leucine-repeat proteins (seeWO 02/20565 and WO 06/083275).

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, patents, published and non-published patentapplications cited throughout this application are expresslyincorporated herein by reference in their entireties.

Example 1: Generation of scFvs with Improved Solubility

In this example, a structural modeling and sequence analysis basedapproach was used to identify mutations in scFv framework regions thatresult in improved solubility.

a) Structural Analysis

The 3D structure of the ESBA105 scFv was modeled using the automatedprotein structure homology-modeling server, accessible via the ExPASyweb server. The structure was analyzed according to the relative surfaceaccessible to the solvent (rSAS) and residues were classified asfollows: (1) Exposed for residues showing a rSAS≥50%; and (2) partiallyexposed for residues with a 50%≤rSAS≥25%. Hydrophobic residues with anrSAS≥25% were considered as hydrophobic patches. To validate the solventaccessible area of each hydrophobic patch found, calculations were donefrom 27 PDB files with high homology to ESBA105 and a resolution higherthan 2.7 Å. The average rSAS and standard deviation were calculated forthe hydrophobic patches and examined in detail for each of them (seeTable 3).

TABLE 3 Assessment of the hydrophobic patches. Surface exposed to theVH/ solvent STDE Sequence Antigen VH/VL VH/CH Residue Domain % % rSASVariability Interface Interface Interface 2 VH 23.06 19.26 10-25%10-25% >0-20% >0-20% 0 4 VH 0.66 1.26  0-10%  0-10% 0 0 5 VH 61.85 12.9650-75% 10-25% 0 >0-20% 0 12 VH 70.27 9.17 50-75% 10-25% 0 0 60-80% 103VH 35.85 5.85 25-50% 10-25% 0  >0-2%  >0-2% 144 VH 62.17 7.82 50-75%10-25% 0 0  >0-2% 15 VL 49.59 9.77 25-50% 10-25% 0 0 0 147 VL 31.1923.32 25-50% 10-25% 0 0 60-80% Column 1, residue position in AHo'snumbering system. Column 2, Domain for the position indicated incolumn 1. Column 3, Average solvent accessible area calculations from 27PDB files. Column 4, Standard deviations of column 3. Columns 5 to 9,Structural role of the hydrophobic patches retrieved from AHo's.

Most of the hydrophobic patches identified in ESBA105 corresponded tothe variable-constant domain (VH/CH) interface. This correlated withprevious findings of solvent exposed hydrophobic residues in an scFvformat (Nieba et al., 1997). Two of the hydrophobic patches (VH 2 and VH5) also contributed to the VL-VH interaction and were therefore excludedfrom subsequent analysis.

b) Design of Solubility Mutations

A total of 122 VL and 137 VH sequences were retrieved from AnnemarieHonegger's antibody web-page (www.bioc.uzh.ch/antibody). The sequencesoriginally corresponded to 393 antibody structures in Fv or Fab formatextracted from the Protein Data Bank (PDB)(www.rcsb.org/pdb/home/home.do). Sequences were used for the analysisregardless of species or subgroup in order to increase the probabilityof finding alternative amino acids with higher hydrophilicity than thenative residue. Sequences having more than 95% identity to any othersequence within the database were excluded to reduce bias. The sequenceswere aligned and analyzed for residues frequency. Sequence analysistools and algorithms were applied to identify and select hydrophilicmutations to disrupt the hydrophobic patches in ESBA105. The sequenceswere aligned following AHo's numbering system for immunoglobulinvariable domain (Honegger and Pluckthun 2001). The analysis wasconstrained to the framework regions.

The residues frequency, f(r), for each position, i, in the customizeddatabase was calculated by the number of times that particular residueis observed within the data set divided by the total number ofsequences. In a first step, the frequency of occurrence of the differentamino-acids was calculated for each hydrophobic patch. The residuefrequency for each hydrophobic patch identified in ESBA105 was analyzedfrom the customized database described above. Table 4 reports theresidue frequency at the hydrophobic patches divided by the totality ofthe residues present in the database.

TABLE 4 Residue frequency of 259 sequences from mature antibodies in anscFv or Fab format for the hydrophobic patches identified in ESBA105.relative frequency in VH relative frequency in VL Residue VH 4 VH 12 VH103 VH 144 VL 15 VL 147 A 0.23046215 0 0 0 3.8647343 0.176821923 C 0 0 00 0 0 D 0 0 0 0 0 0 E 0 0 0 0 0 0 F 0.483091787 0 0.483091787 0 0 0 G 00 0 0 0 0 H 0 0 0 0 0 0 I 0 2.415458937 9.661835749 0 5.31400966270.38834951 K 0 0 0 0 0 0 L 96.61835749 89.85507246 7.24637681227.0531401 45.89371981 15.53398058 M 0 0 10.62801932 1.93236715 00.970873786 N 0 0 0 0 0 0 P 0.966183575 0 0 0.966183575 21.739130430.485436893 Q 0 0 0 0.483091787 0 0 R 0 0 7.246376812 0 0 0 S 00.966183575 0 18.84057971 0 0 T 0 0 15.4589372 50.72463768 0.966183575 0V 1.93236715 6.763285024 49.27536232 0 22.22222222 12.62135922 W 0 0 0 00 0 Y 0 0 0 0 0 0

In the second step the frequency of hydrophilic residues at thehydrophobic patches was used to design the solubility mutations byselecting the most abundant hydrophilic residue at each hydrophobicpatch. Table 5 reports the solubility mutants identified using thisapproach. The hydrophobicity of the parental and mutant residues werecalculated as average hydrophobicity of values published in severalpapers and expressed in function of the level of exposure of the sidechain to the solvent.

TABLE 5 Different solubility mutations introduced in ESBA105 to disruptthe hydrophobic patches Surface Hydo- exposed phobicity Hydo- to the ofphobicity solvent Parental parental Solubility of Residue Domain %residue residue mutation mutations  4 VH 0.66 L 85.2 A 42.7 12 VH 70.27V 73.2 S 28 103  VH 35.85 V 73.2 T 32.8 144* VH 62.17 V 73.2 S 28 15 VL49.59 V 73.2 T 32.8 147  VL 31.19 L 85.2 A 42.7 *The hydrophobic patchat position 144 was exchanged not by the most abundant hydrophilicresidue in the database but for Ser since this was already contained inthe CDR's donor of ESBA105. Column 1, residue position in AHo'snumbering system. Column 2, Domain for the position indicated incolumn 1. Column 3, Average solvent accessible area calculations from 27PDB files. Column 4, parental residues in ESBA105. Column 5, Averagehydrophobicities of column 4, retrieved from AHo's. Columns 6, Mostabundant hydrophilic residue at the position indicated in column 1.Average hydrophobicity of column 6 retrieved from AHo's.

c) Testing of Solubility ESBA105 Variants

The solubility mutations were introduced alone or in multiplecombinations and tested for refolding yield, expression, activity andstability and aggregation patterns. Table 6 shows the variouscombinations of solubility mutations introduced in each ESBA105optimized variant based on potential contribution to solubility and thelevel of risk that the mutation would alter antigen binding.

TABLE 6 Design of solubility variants for ESBA105. Hydrophobic Mutants**surface Parental Opt Opt Opt Opt residue Domain residue 1_0 0_2 1_2 2_415 VL V X X X 147* VL V X  4* VH L X 12 VH V X X X 103* VH V X 144  VH LX X X  *Tested separately in a second round **The underscore separatesthe number of mutations contained in the light and the heavy chainrespectively. Column 1, residue position in AHo's numbering system.Column 2, Domain for the position indicated in column 1. Column 3,Parental residue in ESBA105 at the different hydrophobic patches. Column4, Different variants containing solubility mutations at the positionsindicated,

d) Solubility Measurements

Maximal solubilities of ESBA105 and variants were determined by the PEGprecipitation method as initially described by Atha et al. (JBC, 256:12108-12117 (1981)). In this method, the protein concentration in thesupernatants of centrifugated PEG-protein mixtures is measured andlogarithmically plotted against the PEG concentration. Protein solutionof 20 mg/ml was mixed 1:1 with PEG solutions ranging from 30 to 50%saturation. These conditions were chosen based on the solubility profileobserved for the wild-type ESBA105 after empirical determination oflinear dependence of Log S versus Peg concentration (% w/v). Solubilitycurves of several examples of variant ESBA105 that exhibited superiorsolubility are depicted in FIG. 1. A complete list of solubility valuesis also provided in Table 7.

TABLE 7 Estimated maximal solubility and activity of the mutants incomparison with the parental ESBA105. E105 E105 E105 E 105 VH E105 VLMolecule E105 Opt1_0 Opt0_2 Opt1_2 V103T V147A INTERCERPT 1,956 2,2282,179 2,163 2,223 2,047 Maximal solubility 90.36 169.04 151.01 145.55167.11 111.43 Activity relative to 1 1.4 1.5 1.5 1.2 2 ESBA105

e) Thermostability Measurements

Thermostability measurements for the parental ESBA105 and the solubilityfollow ups were performed using FT-IR ATR spectroscopy. The moleculeswere thermochallenged to a broad range of temperatures (25 to 95° C.).The denaturation profile was obtained by applying a Fouriertransformation to the interferogram signals (see FIG. 2). Thedenaturation profiles were used to approximate midpoints of the thermalunfolding transitions (TM) for every ESBA105 variant applying theBoltzmann sigmoidal model (Table 8).

TABLE 8 Midpoints of the thermal unfolding transitions (TM) for everysolubility variant. ESBA105 E105 Opt1.0 E105 Opt1.2 E105 Opt0.2 E105 VHV103T E105 VL V147A Boltzmann sigmoidal Best-fit values BOTTOM 0.3604−0.405 0.7032 0.4516 0.4691 −0.6873 TOP 100.4 99.3 98.84 99.04 99.299.16 V50 61.53 59.91 59.39 60.86 62.08 55.89 SLOPE 2.935 2.886 3.1172.667 2.682 3.551 Std. Error BOTTOM 0.5206 0.3471 0.6652 0.4953 0.39380.4754 TOP 0.5361 0.3266 0.6116 0.4891 0.4167 0.3714 V50 0.1047 0.066580.1328 0.0949 0.07811 0.0919 SLOPE 0.09039 0.05744 0.1146 0.081990.06751 0.08235 95% Confidence Intervals BOTTOM −0.7432 to 1.464  −1.141to 0.3309 −0.7071 to 2.114  −0.5984 to 1.502  −0.3658 to 1.304  −1.695to 0.3206 TOP   99.25 to 101.5 98.61 to 99.99   97.54 to 100.1   98.01to 100.1   98.32 to 100.1 98.38 to 99.95 V50   61.31 to 61.75 59.77 to60.06   59.11 to 59.67   60.66 to 61.06   61.91 to 62.24 55.70 to 56.09SLOPE   2.743 to 3.127 2.764 to 3.007   2.874 to 3.360   2.494 to 2.841  2.539 to 2.825 3.376 to 3.725 Goodness of Fit Degrees of Freedom 16 1616 16 16 16 R² 0.9993 0.9997 0.999 0.9994 0.9996 0.9996 Absolute Sum26.18 10.8 37.2 24 16.14 15.11 of Squares Sy.x 1.279 0.8217 1.525 1.2251.004 0.9719

iii. Aggregation Measurements

ESBA105 and its solubility variants were also analyzed on atime-dependent test to assess degradation and aggregation behavior. Forthis purpose soluble proteins (20 mg/ml) were incubated at an elevatedtemperature (40° C.) in phosphate buffers at pH6.5. Control samples werekept at −80° C. The samples were analyzed after an incubation period oftwo weeks for degradation (SDS-PAGE) and aggregation (SEC). This allowedfor the discarding of variants that were prone to degradation (see FIG.3) or which exhibited a tendency to form soluble or insoluble aggregates(see Table 9).

TABLE 9 Insoluble aggregation measurements. Protein Protein loss(Insoluble aggregates) ESBA105 0-10% ESBA105 Opt 1_0 0-10% ESBA105 Opt0_2 0-10% ESBA105 Opt 1_2 45-50%  ESBA105 VH V103T 0-10%

iv. Expression and Refolding of Solubility Variants

The solubility mutants were also tested for expression and refoldingyield relative to the parent ESBA105 molecule. The results of thesestudies are shown in Table 10.

TABLE 10 Expression and refolding of solubility variants. ExpressionHydrophobic surface residue relative. to Refolding VH VL ESBA105 Yieldmg/L ESBA105 L4 V12 V103 L144 V15 F52 V147 1.0 34 Opt 1_0 T 1.15 12.5Opt 0_2 S S 1.10 35 Opt 1_2 S S T 0.96 44 Opt 2_4 A S T S T A 1.20 notproducible VH L4A 1.0 not producible VH T 1.1 55 V103T VL A 1.2 20 V147A

Although all the hydrophilic solubility mutants exhibited improvedsolubility in comparison to the parental ESBA105 molecule, only some ofthese molecules exhibited suitable for other biophysical properties. Forexample, many variants had a reduced thermostability and/or refoldingyield relative to the parental ESBA105 molecule. In particular,hydrophilic replacement at position V_(L)147 severely diminishedstability. Solubility mutations that did not significantly affectthermal stability were therefore combined and subjected to furtherthermal stress to confirm their properties.

Three mutants containing a combination of four different solubilitymutations (Opt1.0, Opt0.2 and V_(H):V103T) significantly improved thesolubility of ESBA105 without affecting reproducibility, activity orthermal stability. However, a mutant having the combined mutations ofOpt1.0 and Opt0.2 in ESBA105 (Opt 1_2) exhibited an increased amount ofinsoluble aggregates after incubation for 2 weeks at 40° C. (see Table9). This might be explained by the role of the Val at position V_(L) 15in a beta sheet turn, since Val has the greatest beta sheet propensityof all amino acid. This result demonstrated that a single solubilitymutation at position VL 15 is tolerated, but not in combination withsolubility mutants that disrupt other hydrophobic patches. Therefore,the mutations contained in Opt0_2 and VH:V103T were selected as bestperformers to improve solubility properties of scFv molecules.

Example 2: Generation of scFvs Having Enhanced Solubility and Stability

ESBA105 variants identified by solubility design were further optimizedby substitution with stabilizing mutations identified by Quality Control(QC) assay. A total of 4 constructs were created which contained between1 and 3 of the solubility mutations identified in Example 1 above, incombination with all stabilizing mutations found in QC 7.1 and 15.2(i.e., D31N and V83E in the VL domain and V78A, K43 and F67L in the VHdomain). All optimized constructs yielded more soluble protein than awild-type scFv (see Table 11). The best construct consistently exhibiteda greater than 2-fold increase in solubility over wild-type. Neither theactivity nor the stability of the scFv molecules was significantlyimpacted by the combination of stabilizing and solubility enhancingmutations.

TABLE 11 ScFvs with optimized solubility and stability FTIR PEG ActivityTm solubility relative Protein VL/VH Mutations (° C.) (mg/ml) to E105 kDQC7.1D-N- VL: D31N; V83E 69.0 90 1.7 9.06 × 15.2 VH: V78A; K43R; 10⁻¹⁰F67L QC7.1D-N- VL: D31N; V83E 68.9 106 1.5 8.79 × 15.2 VH VH: V78A;K43R; 10⁻¹⁰ V103T F67L; V103T QC7.1D-N- VL: D31N; V83E 66.6 121 1.2 8.12× 15.2 Opt VH: V12S; V78A; 10⁻¹⁰ 02 K43R; F67L; L144S QC7.1D-N- VL:D31N; V83E 67.3 186 1.5 1.34 × 15.2 VH VH: V12S; V78A; 10⁻⁹ V103T OptK43R; F67L; 0_2 V103T; L144S

The solubility values for all 4 variants were used to deconvolute thecontribution of each mutation to the solubility of the scFv. Allmutations appeared to contribute to the solubility of the scFv in anadditive manner even though several of these residues are relativelyclose to one another both in primary sequence and within the 3Dstructure. The analysis indicated that a combination of threesolubility-enhancing mutations in the VH domain (V12S, L144S, V103T (orV103S)) account for ˜60% of scFv solubility. Since hydrophobic patchesare conserved in the variable domains of all immunobinders, this optimalcombination of mutations can be used to improve the solubility ofvirtually any scFv or other immunobinder molecule.

Example 3: Solubility Optimized Variant of Epi43max, a Potent TNFαBinder

Table 12 depicts characterization data for three optimized variants ofEpi43max, a potent TNF binder. EP43_maxDHP is a solubility enhancedvariant of EP43max and comprises the three solubility enhancingmutations above (V→S at AHo position 12, V→T at AHo position 103, andL→T at AHo position 144). EP43_maxmin and EP43_minmax variants weregenerated by domain shuffling between “min” and “max” grafts. Inparticular, the “minmax” variant comprises the minimal graft (CDR-graftonly) version of the light chain and maximal graft version of the heavychain (i.e., grafted rabbit CDRs plus rabbit framework residues involvedin antigen binding). Conversely, the “maxmin” variant comprised themaximal graft version of the light chain and the minimal graft versionof the heavy chain. The thermal denaturation curves of EP43max and itsoptimized variants were compared by FTIR analysis (see FIG. 4).Epi43minmax was found to have a lower midpoint of unfolding thatEpi43max. Moreover, the mimax variant exhibited a step unfoldingtransition, indicating the both domains unfold at very similartemperatures.

TABLE 12 Biophysical characterization data for three optimized variantsof Epi43max, a potent TNF binder. FT-IR stability RF Refolding FW L929*Kon Kaff KD tm° C. yield Expression screening Purification EP43_max 1.46.4 2.28E+05 5.68E−05 2.49E−10 74.32 21.73 +++ Ok Ok EP43_maxDHP 1.4 6.72.35E+05 2.73E−05 1.16E−10 60.15 17 +++ Ok Ok EP43_maxmin 1.4 Inactive1.46E+05 5.33E−03 3.66E−08 51.76 11 +++ Ok Ok EP43_minmax 1.4 1.62.28E+05 1.98E−04 8.68E−10 65.81 46 +++ Ok ok *L929 [EC50-E105/EC50-X],compared in mass units [ng/ml]

Example 4: Solubility Enhanced Derivatives of VEGF Immunobinders

In addition to the TNF immunobinders described above (ESBA105 andEpi43max), several solubility derivates of VEGF immunobinders wereengineered according to the methods of the invention. In particular,these VEGF immunobinder variants were engineered to have a disruptedhydrophobic patch (“DHP”) by selecting for the following residues: (a)Serine (S) at heavy chain amino acid position 12; (b) Serine (S) orThreonine (T) at heavy chain amino acid position 103; and (c) Serine (S)or Threonine (T) at heavy chain amino acid position 144. The biophysicalcharacteristics of these DHP variants were compared to their wild-typecounterparts. These characteristics included melting temperature or Tm(as determined by Bio-ATR), the percentage of β-sheet loss 60° C. (asdetermined by AquaSpec), the percentage of protein loss followingprecipitation at 60° C., solubility by ammonium sulfate precipitation,refolding yield in production and expression levels in E. coli.

As depicted in Table 14 below, the solubility of one of the VEGFimmunobinders (ESBA578minmax FW1.4 DHP) was significantly enhanced byintroduction of the DHP motif. Moreover, other biophysical properties(e.g., thermal stability) were either not adversely affected or improvedby introduction of the motif. The thermal stability curves used todetermine melting temperature for 578 min-max and its solubilityoptimized variant (578 min-max_DHP) are depicted in FIG. 5 and Table 13,while the ammonium sulfate precipitation curves used to determinesolubility values are depicted in FIG. 6.

TABLE 12 Epi43maxDHP Epi43maxmin Epi43max Epi43minmax (941) (959) (676)(958) V50 60.15 65.81 77.78 51.76 SLOPE 2.618 2.908 10.43 4.297 R²0.9974 0.9969 0.9855 0.9936

TABLE 14 Biophysical Characterization of VEGF Immunobinders SolubilityExpression [EC50 in % Refolding Level TM % β Sheet % Protein ofNH₄(SO₄)₂ Yield (arbitrary Immunobinder [° C.] Loss Loss saturation](mg/L) units) 578-max 70.36 −1.93% 16.20% 27.24 12.5 + 578-max ND ND NDND 11.6 + FW1.4_DHP 578-min-max 71.12 −0.52% 10.99% 28.13 23.93 +++578-min-max 70.18 −0.15% 14.82% 32.36 50.5 +++ FW1.4_DHP

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An immunobinder comprising one of the following solubility enhancingmotifs in the heavy chain amino acid positions 12, 103 and 144 (AHonumbering): (a) Serine (S) at heavy chain amino acid position 12; Serine(S) at heavy chain amino acid position 103; and Threonine (T) at heavychain amino acid position 144; or (b) Serine (S) at heavy chain aminoacid position 12; Threonine (T) at heavy chain amino acid position 103;and Serine (S) at heavy chain amino acid position 144; or (c) Serine (S)at heavy chain amino acid position 12; Threonine (T) at heavy chainamino acid position 103; and Threonine (T) at heavy chain amino acidposition
 144. 2. The immunobinder of claim 1, further comprising: (a)Aspartic acid (D) at light chain amino acid position 31; (b) Glutamicacid (E) at light chain amino acid position 83; (c) Arginine (R) atheavy chain amino acid position 43; (d) Leucine (L) at heavy chain aminoacid position 67; and/or (e) Alanine (A) at heavy chain amino acidposition
 78. 3. A method of enhancing the solubility of an immunobinderin native state, the immunobinder comprising a heavy chain variable(V_(H)) region, or fragment thereof, the method comprising: (a)selecting at least two amino acid positions within the V_(H) region formutation; and (b) mutating the at least two amino acid positionsselected for mutation, wherein the at least two amino acid positions areselected from the group of heavy chain amino acid positions consistingof 12, 103 and 144 (according to AHo numbering convention) and themutating comprises the substitution of the amino acid at the selectedamino acid position against a hydrophilic amino acid.
 4. The method ofclaim 3, wherein the hydrophilic amino acid is (a) Serine (S) at heavychain amino acid position 12; (b) Serine (S) or Threonine (T) at heavychain amino acid position 103; and/or (c) Serine (S) or Threonine (T) atheavy chain amino acid position
 144. 5. The method of claim 3, whereinthe amino acid at the amino acid position selected for mutation is ahydrophobic amino acid.
 6. The method of claim 5, wherein thehydrophobic amino acid is leucine (L) or Valine (V).
 7. The method ofclaim 3, wherein the amino acid selected for mutation of (a) the aminoacid at heavy chain amino acid position 12 is Valine (V); (b) the aminoacid at heavy chain amino acid position 103 is Valine (V); and (c) theamino acid at heavy chain amino acid position 144 is Leucine (L).
 8. Themethod of claim 3, wherein thermal stability, refolding, expressionyield, aggregation and/or binding activity of the immunobinder is notadversely affected by the mutating.
 9. The method of claim 3, whereinthe mutating results in at least a 2-fold increase in solubility. 10.The method of claim 3, wherein the mutating further comprises the stepof introducing one or more mutations at an amino acid position (AHonumbering convention) selected from the group consisting of: (a)Aspartic acid (D) at light chain amino acid position 31; (b) Glutamicacid (E) at light chain amino acid position 83; (c) Arginine (R) atheavy chain amino acid position 43; (d) Leucine (L) at heavy chain aminoacid position 67; and (e) Alanine (A) at heavy chain amino acid position78.
 11. An immunobinder prepared according to the method of claim
 3. 12.The immunobinder of claim 1, which is an scFv antibody, a full-lengthimmunoglobulin, a Fab fragment, a Dab or a Nanobody.
 13. Theimmunobinder of claim 11, which is an scFv antibody, a full-lengthimmunoglobulin, a Fab fragment, a Dab or a Nanobody.
 14. A compositioncomprising the immunobinder of claim 1 and a pharmaceutically acceptablecarrier.
 15. A composition comprising the immunobinder of claim 11 and apharmaceutically acceptable carrier.